scifi-ATAC-seq: massive-scale single-cell chromatin accessibility sequencing using combinatorial fluidic indexing

Single-cell ATAC-seq has emerged as a powerful approach for revealing candidate cis-regulatory elements genome-wide at cell-type resolution. However, current single-cell methods suffer from limited throughput and high costs. Here, we present a novel technique called scifi-ATAC-seq, single-cell combinatorial fluidic indexing ATAC-sequencing, which combines a barcoded Tn5 pre-indexing step with droplet-based single-cell ATAC-seq using the 10X Genomics platform. With scifi-ATAC-seq, up to 200,000 nuclei across multiple samples can be indexed in a single emulsion reaction, representing an approximately 20-fold increase in throughput compared to the standard 10X Genomics workflow. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-024-03235-5.

3. Incubate the PCR strips at 25 °C for 1 h to assemble the Tn5.
4. The assembled Tn5 can be used immediately, or stored at -20 °C.
5. Distribute 1.5 μL Tn5 with adapter B1-B8 to each column of the PCR 96-well reaction plate on ice.6.Similarly, distribute 1.5 μL Tn5 with adapter A1-12 to each row of the PCR 96 well reaction plate on ice.7.Each well now contains 3 μL of well assembled Tn5 with distinct index combinations.
8. The plates can be used immediately or sealed for overnight temporary storage at -20 °C.
5. Approximately 3-4 maize seedlings (7-10 day old) were placed on a petri dish on ice and saturated with 500 mL of chilled NIB-cutting buffer.
6. Using a sterilized single-edge razor blade, seedlings were chopped for about 2 min.to break the cell wall, lyse the chloroplasts and mitochondria, and release nuclei into solution.
8. Pellet the nuclei by centrifugation (swinging-bucket centrifuge rotor) at 500 rcf for 5 min.at 4°C. 9. Remove supernatant carefully and leave some of the supernatant behind if necessary.
11. Slowly add the nuclei suspension on the top of 1 mL 35% percoll buffer (mixed 350 μL Percoll and 650 μL NIB wash buffer).
12. Centrifuge at 500 rcf for 10 min.at 4°C.This step further removes the chloroplasts, mitochondria, and small debris.
13. Slowly remove supernatant from the top to the bottom, and leave about 10 μL of supernatant to avoid removing any nuclei.
15. Remove the supernatant and resuspend in 30 μL TAPS buffer.
16. Take 5 μL nuclei to a centrifuge tube, dilute 10 fold with TAPS buffer, add 0.2 μL DAPI(1 mg/mL).Load 5 μL diluted nuclei to a hemocytometer and check the nuclei quality and density under microscope.
In theory, it is feasible to mix up to 96 samples in one assay, given the use of 96 barcodes in this study.However, in practice, there is a risk of nuclei degradation if they remain in the buffer for an extended period.The nuclei isolation for each sample takes approximately 4 minutes, and an additional 30 minutes are needed for nuclei purification for all samples.In the 8-sample scifi-ATAC-seq, we aim to complete the entire nuclei preparation within 1 hour to minimize the risk of nuclei degradation during the process.Including more samples is possible if a sufficient number of high-quality nuclei can be obtained in the given time frame.From the 8-sample scifi-ATACseq with a 300k input, we obtained about 120k nuclei following strict QC.Therefore, it should be feasible to include up to 12 samples in the 300k input scifi-ATAC-seq assay, ensuring an average of 10k nuclei per sample, which is generally acceptable for most experiments.In animals, a similar approach called txci-ATAC-seq has been developed.It provides a protocol for preparing scATACseq with frozen nuclei, offering the potential to profile up to 96 samples simultaneously [39].
3. Maximum numbers of nuclei in one scifi-ATAC-seq assay: The maximum number of nuclei is primarily determined by the number of unique barcodes, with more indexes reducing the probability of barcode collision when multiple nuclei are present in one droplet.In other words, in the 96-barcodes experiment, the probability of barcode collision in the droplet with a certain number of nuclei is expected to be similar, regardless of the total number of loaded nuclei.To verify this, we compared barcode collision rates for droplets containing one to twenty nuclei in both 100k and 200k input scifiATAC-seq.The results showed an increase in the barcode collision rate with more nuclei in the droplet, yet showing similarity between 100k and 200k input scifi-ATAC-seq, where the collision rate is approximately 15% for droplets with 10 nuclei (Additional file 1: Fig. S9b).However, the distribution of the number of nuclei in droplets for 100k, 200k, and 300k input nuclei is not a normal distribution but a biased distribution, with most droplets containing one to ten nuclei (Additional file 1: Fig. S1d,f; Additional file 1: Fig. S6c).Therefore, it is possible to further decrease the barcode collision rate by removing droplets with a high number of nuclei.For instance, the barcode collision rate could be reduced to about 4% and 6% when filtering droplets with more than 10 nuclei while retaining over 90% and 80% of the total non-collision nuclei for 100k and 200k input scifi-ATAC-seq, respectively (Additional file 1: Fig. S9c-e).
In one assay from the 10X Genomics Chromium Controller, approximately 70k droplets were generated, and about 90% of these droplets could be filled with 383k input nuclei (Additional file 1: Fig. S9a).However, as the number of input nuclei increases, there is a higher risk of chip clogging due to the high density of nuclei.We believe it might be feasible to load 400k nuclei in each run and selectively retain only the droplets with no more than 10 nuclei for downstream analysis using the 96-barcodes scifi-ATAC-seq method.

Potential limits for mixing samples from different species in one assay:
There is a potential for sequencing throughput bias among nuclei from different species if they contain significantly different sizes of chromatin accessibility regions due to variations in genome size and as well as GC content variation among species.

Tn5 adapters 1 .
Dissolve 12 Tn5-ME-Ax and 18 Tn5-ME-Bx with the complementary Tn5-ME-Rev single stranded DNA oligonucleotides (oligos) with TE to 200 μM.2. Mix equal volume of Tn5-ME-Ax and Tn5-ME-Rev in a PCR tube to obtain a 100 μM adapter stock.3. Similarly, mix equal volume of Tn5-ME-Bx and Tn5-ME-Rev.4. Heat the oligo mixture to 98°C in a PCR instrument for 2 min.and gradually lower the temperature to 25°C by cycling (-1°C per 10 sec cycle).